Evidence that the use of positive inotropic agents was associated with an increase in mortality led, in the early 1980s, to a radical change in the approach to chronic heart failure (HF), which switched to the prevention of the maladaptive response (also known as myocardial remodelling) triggered by an initial contractile deficit.1 Since then, the use of inotropic agents has been confined to the acute phases of the disease or whenever the patient’s haemodynamics are severely compromised by pump failure. Nonetheless, it is tempting to speculate that if contractile deficit could be safely countered at its early stage, (mal)adaptive neurohumoral responses would be minimised. Notably, current therapeutic approaches mostly antagonise the cellular effects of such responses, including their adaptive component. This conceivably reduces cardiac adaptability to haemodynamic perturbations, potentially with a negative impact on disease evolution (e.g. by increasing the volumes required to support an adequate cardiac output). This has prompted the search for novel strategies to achieve positive inotropy without the undesired effects, to enable long-term use.
Our involvement in the development of SERCA2a-activating drugs started in 2001, when we were asked by a corporate research laboratory (Prassis-Sigma Tau) to provide a mechanistic interpretation of the surprisingly favourable therapeutic profile (inotropy/toxicity) of a molecule, PST2744, then in their pipeline as a ‘digitalis-like’ compound.2 In this brief review, we will revisit the discovery of the unforeseen properties of PST2744, later renamed istaroxime, that are likely to account for its desirable profile, and our journey toward the design and validation of derivatives with improved properties.3 Because of the novelty of its mechanism of action, PST2744 is a first-in-class agent; indeed, it is the first small-molecule tool for stimulation of sarcoplasmic reticulum (SR) Ca2+ ATPase isoform 2a (SERCA2a).4
Rationale for SERCA2a Stimulation
SERCA2a is a Ca2+-ATPase located in the membrane of the major intracellular Ca2+ store (the SR). Its activity clears approximately 70% of the Ca2+ involved in excitation–contraction coupling (Ca2+ transient) from the cytosol, and entirely accounts for Ca2+ uptake by the SR (internally recycled component of the transient). As such, SERCA2a plays a pivotal role in muscle contraction and relaxation; therefore, its modulation is relevant to inotropy and lusitropy. Partial SERCA2a inhibition by phospholamban (PLN) under basal conditions generates a functional reserve that can be recruited by adrenergic activation through protein kinase A (PKA)-mediated PLN phosphorylation, which reduces PLN affinity for SERCA2a.5
SERCA2a activity may be severely depressed in HF, mostly because of defective PLN phosphorylation, sometimes associated with downregulation of SERCA2a expression.6 The concept of therapeutic SERCA2a enhancement in HF has already been explored in a large gene-therapy study.7 The disappointing outcome of that study is most likely to be due to the low transduction efficiency of the viral vectors in a non-selected population, in whom significant titres of antiviral antibodies are to be expected. The availability of small-molecule SERCA2a stimulators, delivered as normal drugs, would circumvent this problem and the exorbitant costs of gene therapy.
While compensatory mechanisms tend to minimise the effect of SERCA2a dysfunction on force development, this occurs at the expense of altered Ca2+ distribution in the intracellular compartments. In other words, even when contractile force can be maintained, higher cytosolic Ca2+ levels are required to achieve it if SERCA2a is depressed. This is best summarised by the term ‘Ca2+ decompartmentation’, a condition in which Ca2+ localisation is lost, and electrical and mechanical performance is destabilised. Because of the lower energy expenditure of SR Ca2+ recycling, Ca2+ decompartmentation may also reduce the thermodynamic efficiency of excitation–contraction coupling, as indicated by the effect of SERCA2a stimulation on the cell energy content of diseased myocytes.8–10 Persistently elevated cytosolic Ca2+ levels should also play a pivotal role in myocardial remodelling, as suggested by its reversal by constitutive SERCA2a stimulation.11,12 However, it has been reported that SERCA2a stimulation fails to prevent activation of the hypertrophy-triggering calcineurin–nuclear factor of activated T-cells pathway, and that a mutation causing marked PLN loss of function (i.e. SERCA2a stimulation) is associated with myocardial remodelling in humans.13,14 Therefore, whether SERCA2a stimulation may prevent myocardial remodelling is still a matter of debate; differences in the extent of such stimulation and/or ancillary effects (toxicity) of mutant PLN proteins might possibly contribute to the contrasting results.
SERCA2a activation is an important component of myocardial response to catecholamines (and to drugs enhancing cAMP–PKA signalling), which is precisely what β-blocker therapy aims to prevent. Thus, SERCA2a stimulation might appear irrational in the context of HF; nonetheless, this view is probably simplistic. cAMP–PKA signalling simultaneously affects multiple elements of the excitation–contraction machinery, resulting in upregulation of intracellular Ca2+ content, electrical instability and hypertrophic gene transcription. In contrast, the main action expected from SERCA2a stimulation alone is to promote Ca2+ confinement inside the SR, thus decreasing the cell Ca2+ content required to achieve a certain force development.15 This action may be particularly beneficial in the context of HF, in which SERCA2a depression is a major pathogenetic mechanism. Although Na+/K+ pump inhibition may also increase overall cell Ca2+ content, it does not promote Ca2+ confinement inside the SR and this probably accounts for its proarrhythmic potential.15
Overall, restoration of SERCA2a activity seems highly desirable in HF, with expected inotropic, lusitropic and antiarrhythmic benefits. An important caveat to this view, which generates controversy regarding the benefit of SERCA2a stimulation, is that the resulting increase in SR Ca2+ content may promote random opening of Ca2+–SR channels (i.e. ryanodine receptors, RyRs), thus facilitating spontaneous Ca2+ release events (SCRs) of proarrhythmic significance. The balance between the advantages and disadvantages of SERCA2a stimulation may conceivably depend on the extent of the initial SERCA2a depression (the larger the better) and on the condition leading to it, with the worst cases theoretically represented by those characterised by primary RyR instability (e.g. RyR mutations). For a detailed discussion of this issue, we refer the reader to the review by Zaza and Rocchetti.16 Nonetheless, bench evidence and the clinical observation that SERCA2a stimulation by istaroxime is not associated with the proarrhythmic effect expected from concomitant Na+/K+ pump inhibition indicates that the advantages may prevail in such a balance.17–20
Discovery
PST2744 ((E,Z)-3-((2-aminoethoxy)imino)androstane-6,17-dione hydrochloride) (Figure 1, istaroxime), a derivative of 3,6,17-androstanetrione chemically unrelated to cardiac glycosides, was initially identified as a potential Na+/K+ pump inhibitor in a molecular modelling study proposing a new 3D model for the binding of cassaine (a plant alkaloid) at the digitalis receptor site.21
PST2744 was indeed shown to inhibit the Na+/K+ pump (digitalis-like action), thus expectedly exerting positive inotropy in animal studies (on guinea pigs).2 Nonetheless, it was soon noted that, at comparable inotropic effect, proarrhythmia and overall toxicity were significantly lower with PST2744 than with digoxin, the gold standard for digitalis action.2 Given that proarrhythmia, expected from Na+/K+ pump blockade, provides the main rationale for avoiding chronic use of digitalis, such a serendipitous observation triggered interest. As a research group focused on cellular mechanism of arrhythmogenesis, we were then invited to investigate if and how the electrophysiological effect of PST2744 at the cellular level differed from that of digoxin.
The study of cellular electrophysiology involves measurements in isolated myocytes. Therefore, our first concern was to check whether the lower proarrhythmic effect of PST2744 observed in whole animals, could be reproduced in isolated ventricular myocytes. In guinea pig ventricular myocytes, the threshold for aftercontractions (i.e. the indicator of arrhythmia) was at 20% and 60% of twitch amplitude enhancement (i.e. the indicator of inotropy) with digoxin and PST2744, respectively. Overall, in the low equi-inotropic concentration range, there were significantly fewer aftercontractions with PST2744 than with digoxin, and at concentrations lethal with digoxin, no death occurred with PST2744.2 These encouraging results with PST2744 were further confirmed in dogs with chronic ischaemic HF.22
We then proceeded to an extensive comparison of the electrophysiological effects of PST2744 versus digoxin, tested in guinea pig myocytes at equi-inotropic concentrations (4:1).23 PST2744, but not digoxin, reduced the amplitude of the transient inward current (ITI) following a depolarisation step. This could be explained by either a reduction in the conductance of the Na+/Ca2+ exchanger (NCX), the charge carrier for ITI, or by an acceleration of the Ca2+ transient decay, which would blunt the driving force on NCX. NCX conductance was indeed slightly inhibited by PST2744, but the small magnitude of this effect encouraged us to investigate also the latter hypothesis.
This was pursued in the following study, again comparing equi-inotropic PST2744 and digoxin concentrations in guinea pig myocytes.24 We first evaluated intracellular Ca2+ dynamics under physiological conditions, to find that PST2744 stimulated Ca2+ dynamics more than digoxin. This effect was expected from both agents because of the shared modulation of the Na+/K+ pump; however, the quantitative difference suggested that PST2744 might be endowed with an extra effect. We then repeated the evaluation under conditions ruling out the Na+/K+ pump as the drug target and found that under such conditions PST2744, but not digoxin, robustly enhanced Ca2+ uptake by the SR. At this point, SERCA2a was a strong candidate-target to account for the effect specific of PST2744; therefore, we sought the collaboration of Dr Istvan Jona, expert in the evaluation of SERCA2a function (ATPase activity) in a simplified (cell-free) system (microsomes). As a result of the collaboration, we showed that PST2744 was indeed able to enhance the Ca2+ dependency of SERCA2a activity.24 Albeit combined with Na+/K+ pump blockade in the case of PST2744, this represented an entirely novel action, of potential relevance to the modulation of myocardial mechanical and electrical functions. Notably, by combining the new findings with those highlighting the better safety profile of PST2744, we hypothesised that SERCA2a stimulation might somehow limit the proarrhythmic effect of Na+/K+ pump inhibition. This view, of obvious translational relevance, found strong support in subsequent studies by others, demonstrating that SERCA2a stimulation may prevent proarrhythmia related to SR instability.17,18
Translational Applicability and Conceptual Refinement
Although SERCA2 stimulation may also be beneficial for cell function and viability in non-cardiac tissues, the obvious therapeutic target for a SERCA2a stimulator is HF.25,26 SERCA2a function is generally depressed in remodelled myocardium; hence the concern arose that PST2744 might be unable to enhance SR Ca2+ uptake in failing hearts. To address this concern, we tested PST2744 in a guinea pig model of hypertrophy/HF induced by chronic mechanical overload (trans-aortic constriction).27 As expected from SERCA2a downregulation, SR Ca2+ uptake was severely depressed in remodelled myocytes; nonetheless, our concern was proven unjustified by the evidence that PST2744 enhanced SR Ca2+ uptake significantly more in remodelled than in normal myocytes.27 This observation was reproduced in later studies. For example, in rats (intrinsically endowed with strong SERCA2a) the effects of SERCA2a stimulators on SR Ca2+ uptake became detectable only after SERCA2a function had been depressed (by streptozotocin [STZ]-induced diastolic dysfunction).28 We have recently addressed the mechanism underlying this puzzling finding in a computational study. The (as yet unpublished) results indicate that the effect of SERCA2a stimulation on SR Ca2+ uptake rate is expectedly maximal at intermediate levels of SERCA2a function (Zaza A, unpublished data). This is because at higher levels, the resulting increase in SR Ca2+ content facilitates RyR channel opening, leading to SR Ca2+ leak outweighing Ca2+ uptake by SERCA2a; this is consistent with the concept of strict homeostatic control of cell Ca2+ content.
Homeostatic control of cell Ca2+ content predicts that, unlike the multi-target effects of PKA activation (e.g. by β-adrenergic stimulation), selective SERCA2a stimulation should affect cell Ca2+ content only marginally. If so, what should we expect from SERCA2a stimulators in terms of overall Ca2+ dynamics, inotropy/lusitropy and proarrhythmia? We addressed this question in a study on murine ventricular myocytes, also meant to test for species dependency of istaroxime’s effects.15 We again compared istaroxime with digoxin but focused on the Ca2+ distribution between the SR and cytosolic compartments at a given level of Na+/K+ pump inhibition and inotropy. The results confirmed the difference between the two agents in terms of SR Ca2+ uptake modulation and showed that this difference translated into a diverging compartmentation of intracellular Ca2+ during quiescence and sustained stimulation. More specifically, whereas digoxin caused immediate cytosolic Ca2+ accumulation in quiescent myocytes, istaroxime did not. During sustained stimulation digoxin shifted the Ca2+ transients to higher Ca2+ levels without changing their amplitude (i.e. it increased both systolic and diastolic Ca2+ similarly); istaroxime increased diastolic Ca2+ less than systolic Ca2+, thus achieving a similar Ca2+ transients amplitude but at lower diastolic Ca2+. Finally, istaroxime, but not digoxin, slightly increased SR Ca2+ content.15 This suggests that concomitant SERCA2a stimulation may improve subcellular Ca2+ compartmentation, thus decreasing the levels of diastolic Ca2+ associated with a given inotropy level. Notably, the relationship between SR Ca2+ leak and load was similar between the two agents, thus indicating that the improved Ca2+ compartmentation by istaroxime was due to SERCA2a stimulation only. Besides the predictable consequence on the ratio between inotropic and lusitropic effects, improved Ca2+ compartmentation may have an impact on multiple aspects of myocyte function, including SR stability, energy consumption and, possibly, modulation of hypertrophic transcription.16
From a clinical standpoint, preclinical studies indicate that selective SERCA2a stimulation would provide inotropic and lusitropic effect at a low proarrhythmic risk and, possibly, improve the biology of the failing myocyte. Whereas the ino-lusitropic effect is expectedly smaller than that achieved by PKA activation (e.g. by amine drugs), which entails additional mechanisms, the salient feature of the intervention is probably the ability to restore intracellular Ca2+ compartmentation. This compartmentation may be pivotal in preserving electrical stability and in limiting the evolution of cell damage. The conspicuous lack of a proarrhythmic effect of istaroxime in clinical studies (see below) argues against the concern that enhanced SR Ca2+ uptake (at least at the level achieved) might be detrimental for electrical stability; it even suggests that SERCA2a stimulation may counter digitalis-like arrhythmogenesis (see Rocchetti and Zaza for further discussion16). Clinical data are available only for acute istaroxime treatment; conclusions regarding the long-term risk–benefit ratio of PLN antagonism in the evolution of cardiomyocyte damage (see discussion above) await information from studies on long-term use of SERCA2-selective compounds.
Mechanism of SERCA2a Stimulation
In cardiac, but not skeletal, myocytes, SERCA2a activity is physiologically restrained by an association with PLN, which is, in turn, countered by PLN phosphorylation.5 The hypothesis that istaroxime might stimulate cardiac SERCA2a by preventing such an interaction was first tested in our 2008 study, which showed istaroxime to be inactive in skeletal muscle microsomes devoid of PLN.27 Definitive confirmation that istaroxime may stimulate SERCA2a by PLN antagonism came from a further study in microsomes isolated from various sources, showing that the agent physically disrupted the SERCA2a–PLN interaction and its functional effect could be reinstated by SERCA2a–PLN reconstitution.29 The same study also reported istaroxime effect to be independent of PLN phosphorylation, thus suggesting direct interference with SERCA2a-PLN interaction. PLN antagonism may also account for the persistence of drug efficacy in remodelled myocytes, in which defective PLN phosphorylation (as opposed to reduced SERCA2a expression) prevails in depressing SERCA2a activity.
An istaroxime-induced increase in SERCA2 protein expression was recently described, by immunohistochemistry, in osteocytes of mice with type 2 diabetes.26 In these cells, istaroxime restored the deranged Ca2+ dynamics and, with it, bone mechanoresponsiveness. This was the first study to show an istaroxime mechanism of action upstream of PLN antagonism. Nonetheless, in diabetic osteocytes the restoration of phasic and regulated Ca2+ handling led to an overall improvement of gene transcription efficiency, possibly including the SERCA2 gene.26 Therefore, SERCA2 transcript upregulation might also be secondary to istaroxime-induced increase of SERCA2a function.
Parallel Animal and Clinical Studies
In 2007, a haemodynamic study in dogs with chronic (ischaemic) HF found istaroxime to improve systolic and diastolic function, notably without increasing myocardial O2 consumption.30 The authors of the study attributed the effect to SERCA2a stimulation, but forgot to quote where the notion that istaroxime stimulates SERCA2a came from.24 While apparently incompatible with an increase in cardiac work (implied by positive inotropy), this observation is consistent with the energy-sparing effect of prevailing Ca2+ cycling by SERCA2a, which may improve contraction efficiency (see above).
This preclinical evidence was followed by the evaluation of a 6-hour istaroxime infusion in patients with advanced HF (ejection fraction [EF]<35%).19 In that study istaroxime, although failing to affect EF, improved several haemodynamic parameters, including systolic blood pressure (SBP). This was not associated with changes in vascular resistances, signs of myocardial damage, or proarrhythmia; however, the istaroxime plasma half-life was deceptively short (<1 hour), thus ruling out oral administration as a treatment modality. Again, the authors attributed istaroxime’s effect to SERCA2a stimulation, but to support this view, they surprisingly quoted an article2 published 3 years before the actual discovery of the action.24
A more prolonged (24-hour) istaroxime infusion in HF patients was evaluated in 2020.20 That recent study confirmed istaroxime efficacy, with more clear-cut haemodynamic effects and reduction of biochemical markers of chamber dilation, again in the absence of proarrhythmia or signs of myocardial damage. Blood samples for pharmacokinetic analysis were collected from a subset of patients, but plasma drug levels were not reported.
The hypothesis that inotropy by a non-adrenergic intervention may be safe and effective in haemodynamic support of the subset of HF patients in pre-cardiogenic shock (SBP<90 mmHg) was recently tested. In that pilot multicentre study (SEISMiC trial), 24-hour istaroxime infusion increased SBP and improved several echocardiographic indexes without major side effects.31 Notably, SBP and cardiac index increased concomitantly, an effect unique to istaroxime (i.e. never observed for other IV drugs in this class of patients). Even considering that istaroxime is also endowed with a digitalis-like action, this observation points to the potential benefit of non-adrenergic SERCA2a stimulation. Based on such positive findings, a further trial (SEISMiC-B) of an increased istaroxime infusion (to 60 hours) has been announced.32
Further articles supporting the positive haemodynamic effects of istaroxime in acute HF patients in general were published thereafter, as summarised in two recent meta-analyses.33,34 An exception to this view comes from the failure of istaroxime to improve diastolic indexes in a small sample of patients with HF with preserved EF during exercise.35 Although this observation may seem surprising in light of the primarily lusitropic effect of SERCA2a stimulation, structural components (collagen, titin, etc.) may prevail over dysregulation of Ca2+ handling in hampering relaxation of hypertrophied hearts.36
Development of Istaroxime Derivatives
As noted in the introduction, besides their use in infusions in acute HF, low-toxicity inotropic drugs might be used in the long term for contractile support at early disease stages. This requires repetitive oral dosing at convenient intervals, for which istaroxime is ill-suited because of its very short plasma half-life. A further problem with long-term istaroxime treatment may be the presence of an oxime moiety, which is considered as potentially genotoxic in long-term use. Finding a solution to these two problems was the main motive for our subsequent endeavour to suitably modify the istaroxime molecule. Such an endeavour was carried out through close collaboration with biochemists and synthetic chemists in a departmental joint-laboratory initiative.
Istaroxime is quickly metabolised in the liver by oxidative deamination of the primary amino group and reduction of the carbonyl in position 6, resulting in a terminal metabolite named PST3093. Pharmacokinetic analysis of the human blood samples collected in the 2020 clinical study confirmed the very short half-life of istaroxime, but detected a very large accumulation of PST3093, which soon exceeded the istaroxime plasma levels and decayed with a half-life >8 hours (Figure 1).20,37
This serendipitous observation led us to consider the possibility that PST3093 might retain istaroxime’s effects, thus affording a means to achieve them chronically, by oral dosing. As an experimental model to test this hypothesis we chose the rat; however, preliminary experiments showed that healthy rat myocytes were insensitive even to istaroxime. Therefore, following the idea that depressed SERCA2a is more responsive to stimulators, we switched to a diabetic rat model (STZ-induced diabetes). In diabetic myocytes, SERCA2a activity was indeed severely depressed and PST3093 almost completely recovered it, as well as SR Ca2+ uptake, to the levels observed in healthy myocytes. PST3093 was also found to restore in vivo haemodynamics in the diabetic model, thus demonstrating the pivotal role of SERCA2a downregulation in the dysfunction of diabetic hearts.37 At concentrations corresponding to the observed plasma levels, PST3093 efficacy was similar to that of istaroxime. This suggests that the metabolite might actually account for the haemodynamic effects of the parent compound observed in patients.20 As for istaroxime, SERCA2a modulation by PST3093 was found to depend on the presence of PLN, thus qualifying the compound as a PLN antagonist. We then proceeded to evaluate modulation of the Na+/K+ pump and found that PST3093 did not share the inhibitory effect of istaroxime (Figure 2).37 Except for the persistence of a minor shortening of repolarisation, PST3093 was also found to be devoid of electrophysiological effects and toxicity, consistent with the safety of istaroxime infusion, during which the metabolite was present at high concentrations.20,37 To summarise, at variance with its parent compound, PST3093 was found to be a pure SERCA2a stimulator, with an interesting pharmacodynamic profile, and was an excellent starting point for the design of an entirely novel class of ino-lusitropic agents.
PST3093 still retains the oxime moiety, which would hinder its chronic therapeutic use. To address this problem, we started a collaboration with a synthetic chemistry group in our department, which yielded a series of new PST3093 derivatives devoid of the oxime moiety.38 The oxime C double bond was replaced with either an alkene group, or with a saturated C-C bond in the β configuration. The new molecules were screened for their stimulation of SERCA2a activity in microsomes, highlighting the essential role of the saturated C-C bond in preserving the effect. Two leads were further validated for modulation of SR Ca2+ uptake and electrophysiology in myocytes of diabetic rats and in the same model for in vivo haemodynamic effects.38 Similar to the parent compound, the derivatives failed to block the Na+/K+ pump (pure SERCA2a activators; Figure 2) and their effect depended on the presence of PLN (PLN antagonists). Moreover, a derivative of PST3093 (compound 8), still devoid of a Na+/K+ pump blocking effect (Figure 2), was shown to reverse STZ-induced diastolic dysfunction in vivo by oral administration (Figure 3), thus providing a mechanism-based lusitropic treatment for chronic HF.39
Conclusion
Insightful interpretation of the favourable therapeutic profile of PST2744 (now known as istaroxime), a putative digitalis-like compound, has disclosed the possibility of achieving stimulation of SERCA2a function by a small molecule. This represents a significant advancement with respect to SERCA2a modulation by gene-based approaches, and establishes a novel class of ino-lusitropic drugs. Subsequent refinement of the parent compound has yielded derivatives with strong SERCA2a selectivity and long plasma half-lives, suitable for long-term use. While the safety and haemodynamic efficacy of istaroxime in the acute HF setting have been confirmed by several independent trials, the long-term use of its derivatives remains to be tested in the clinical setting. Intriguingly, evidence of the biological efficacy of istaroxime in extra-cardiac targets is emerging, thus suggesting an unforeseen benefit of SERCA stimulation in restoring the function of multiple organs.26